Bioreduction of toxicity influenced by bioactive molecules secreted under metal stress by Azotobacter chroococcum

Abstract

Heavy metal pollution destruct soil microbial compositions and functions, plant’s performance and subsequently human health. Culturable microbes among many metal abatement strategies are considered inexpensive, viable and environmentally safe. In this study, nitrogen fixing bacterial strain CAZ3 recovered from chilli rhizosphere tolerated 100, 1000 and 1200 µg mL⁻¹ of cadmium, chromium and nickel, respectively and was identified as Azotobacter chroococcum by 16S rDNA sequence analysis. Under metal stress, cellular morphology of A. chroococcum observed under SEM was found distorted and shrinkage of cells was noticed when grown with 50 µg mL⁻¹ of Cd (cell size 1.7 µm) and 100 of µg mL⁻¹ Ni (cell size 1.3 µm) compared to untreated control (cell size 1.8 µm). In the presence of 100 µg mL⁻¹ of Cr, cells became elongated and measured 1.9 µm in size. Location of metals inside the cells was revealed by EDX. A dose dependent growth arrest and consequently the death of A. chroococcum cells was revealed under CLSM. A. chroococcum CAZ3 secreted 320, 353 and 133 µg EPS mL⁻¹ when grown with 100 µg mL⁻¹ each of Cd, Cr and Ni, respectively. The EDX revealed the presence of 0.4, 0.07 and 0.24% of Cd, Cr and Ni, respectively within EPS extracted from metal treated cells. Moreover, a dark brown pigment (melanin) secreted by A. chroococcum cells under metal pressure displayed tremendous metal chelating activity. The EDX spectra of melanin extracted from metal treated cells of A. chroococcum CAZ3 displayed 0.53, 0.22 and 0.12% accumulation of Cd, Cr and Ni, respectively. The FT-IR spectra of EPS and melanin demonstrated stretching vibrations and variations in surface functional groups of bacterial cells. The C-H stretching of CH₃ in fatty acids and CH₂ groups, stretching of N-H bond of proteins and O-H bond of hydroxyl groups caused the shifting of peaks in the EPS spectra. Similar stretching vibrations were recorded in metal treated melanin which involved CHO, alkyl, carboxylate and alkene groups resulting in significant peak shifts. Nuclear magnetic resonance (NMR) spectrum of EPS extracted from A. chroococcum CAZ3 revealed apparent peak signals at 4.717, 9.497, 9.369 and 9.242 ppm. However, ¹H NMR peaks were poorly resolved due largely to the impurity/viscosity of the EPS. The entrapment of metals by EPS and melanin was confirmed by EDX. Also, the induction and excretion of variable amounts of metallothioneins (MTs) by A. chroococcum under metal pressure was interesting. Conclusively, the present findings establish- (i) cellular damage due to Cd, Cr and Ni and (ii) role of EPS, melanin and MTs in adsorption/complexation and concurrently the removal of heavy metals. Considering these, A. chroococcum can be promoted as a promising candidate for supplying N efficiently to plants and protecting plants from metal toxicity while growing under metal stressed environment.